Can$we$eliminate$importantbacterial$pathogens$with ......Human Neisseria meningitidis meningitis,...

Can we eliminate important bacterial pathogens with protein-‐based vaccines?

Engineered antigens derived from bacterial transferrin receptors

Dr. Tony Schryvers (University of Calgary) Vaccine Innovation Conference May 26, 2015

Host-‐Restricted Upper Respiratory Tract Pathogens

Gram-‐Negative Species

• Neisseria meningitidis • Haemophilus influenzae • Moraxella catarrhalis

Gram-‐Positive Species

• Streptococcus pneumoniae

-‐ Responsible for 20-‐25% of deaths < 5 yrs

-‐ Cause of most common bacterial infection

Common Features • highly host-‐adapted & host

specific • URT only reservoir –

transmission by respiratory route

• are naturally transformable • ‘horizontal’ gene transfer

contributes to genetic and antigenic variation

• challenge to develop a broadly protective vaccines

Non-Invasive Invasive

Bacterial Respiratory Tract Pathogens

• Responsible for a spectrum of invasive and non-‐invasive infections

• Colonization precedes infection

• Bacteria causing invasive infection possess polysaccharide capsule

S. pneumoniaeN. meningitidisM. catarrhalisH. influenzae

nontypeable

Sinusitis

Ear Infection

nontypeable

Pneumonia

Meningitis

Pneumonia

type b

type b

type b

Sepsis

0

23

45

68

90

1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

vaccine

Type b H. influenzae (UK) 1992

S. pneumoniae PCV7 – 7 serotypes

(Calgary) 2002

Conjugate Capsular Vaccines

Reduce Infection by Targeted Serotypes

Vaccine serotypes


• Designed to prevent invasive infection • Studies demonstrate that systemic vaccination

results in: – elimination of targeted serotypes from mucosal

carriage – disease reduction in non-vaccinated populations

0%

5%

10%

15%

20%

Spring '03 Spring '04 Spring '0512 months 18 months4.5 years

Carriage % Children – vaccine types

0

15

30

45

60

1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

All SerotypesPCV7 Serotypesnon-PCV7 Serotypes

Invasive Disease Adults > 65


• Highly specific immunity • reduction in carriage by vaccine serotypes coupled

with increase in non-vaccine serotypes

0%

5%

10%

15%

20%

Spring '03 Fall '03 Spring '04 Fall '04 Spring '05 Fall '0512 months 18 months 4.5 years

0%

5%

10%

15%

20%

Spring '03 Spring '04 Spring '05

12 months 18 months4.5 years

% Children – vaccine types % Children – non- vaccine types

Calgary – CASPER


• Highly specific immunity - reduction in disease by vaccine serotypes coupled with increase in non-vaccine serotypes

0

75

150

225

300

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

PCV719AOther Vaccine PreventableNon-Vaccine PreventableUnknown

vaccine

Canada IMPACT


PROS • Effective reduction in disease by strains expressing targeted

polysaccharide capsules • Reduction in colonization led to decreased transmission and reduction in disease in non-‐immunized (herd effect)

CONS • Not effective against other capsular types – may result in disease by other types (capsule switching)

• Not effective for non-‐immunogenic capsular types (group B meningococci)

Development of broadly cross-‐protective vaccines may need to target protein antigens

Iron Homeostasis: Role of Transferrin (Tf)

• Tf shuttles iron within body to cells requiring iron (Fe) • Tf is normally only partially saturated • Tf keeps the level of free Fe below that needed for

growth of pathogens

• Tf is a critical source of iron during invasive infection

• There is limited information regarding iron homeostasis on mucosal surfaces

• Tf known to be critical iron source on the mucosl surface for some bacteria (Neisseria gonorrhoeae, Actinobacillus pleuropneumoniae)

Human Neisseria meningitidis meningitis, sepsis

Neisseria gonorrhoeae gonorrhea

Haemophilus influenzae meningitis, sepsis

Haemophilus influenzae (NT) otitis media, COPD, pneumonia

Moraxella catarrhalis otitis media, COPD, pneumonia

Cattle Sheep Goat Mannheimia haemolytica BRD (shipping fever)

Pasteurella multocida BRD

Histophilus somni BRD, invasive disease

Moraxella bovis pinkeye

Pasteurella trehalosi sepsis / RD

Pig Actinobaccilus pleuropneumoniae pneumonia

Haemophilus parasuis Glasser’s disease (invasive disease & pneumonia)

Actinobacillus suis sepsis, hemorrhagic/embolic lesions of lung

Bacterial Transferrin Receptors

FbpA

TbpA

FeFbpB/C

TonB

ExbBExbD

TbpB

Tf

Receptor-‐mediated iron acquisition from transferrin (Tf)

• Surface lipoprotein,TbpB (transferrin binding protein B) captures Fe loaded Tf

• TbpB transfers Fe-‐Tf to TbpA, an integral outer membrane protein

• TbpA removes Fe from Tf and transports Fe across the outer membrane

• FbpA (ferric binding protein A) shuttles iron to inner membrane complex

• FbpBC inner membrane complex transports Fe into the cell

TbpB Vaccines – A Brief History

1990: patent on Tf/Lf receptors filed

Meningococcal Vaccines: • 1990 Pasteur Merieux (currently Sanofi Aventis) licensed Tbps • 1990-‐1998 native TbpB shown to be immunogenic and protective in mice/rabbits, 3 TbpB variants provide broad coverage

• 1998 human Phase I clinical trial (w/o adjuvant) -‐ disappointing results • 2001 license abandoned

Animal Vaccines: • recombinant proteins tested directly in pigs and cattle Key Relevant Research Findings: • human gonococcal and pig experiments demonstrate Tbps critical for survival and disease

2001 -‐2006: noncompete clause, insufficient funding 2008: • AHFMR Team Grant & sabbatical, structure-‐based vaccine design

TbpB Vaccines - Rationale for Structure-based Antigen Engineering Approach

Hypotheses:

• host Tf binds to TbpB during systemic immunization & interferes with development of optimal protective response • explains poor performance in Phase I trial & only moderate success for animal vaccines

• solution: design non-‐Tf binding antigens

• TbpBs bind to same region of Tf requiring conserved binding interface, antigenic variation involves immunodominant ‘decoy’ B-‐cell epitopes • solution: engineer antigens with enhanced ability to induce cross-‐protective immune response

• remove decoy epitopes and preserve conserved binding interface

barrel barrel

barrel

barrel

handle

handle

C-‐lobe

C-‐lobe

N-‐lobe

N-‐lobe

anchorpeptide

TbpB Structure (porcine pathogen -‐ A. pleuropneumoniae)

Trevor Moraes

Site-‐Directed Mutants

Single mutations designed to reduce/eliminate Tf binding.

Panel A: Targeted residues

Panels B – G: Superposed mutant and

wild-‐type structures

Mutations had no effect on overall structure

Site-‐Directed Mutants

Single mutation results in dramatic loss in Tf binding

Affinity binding constants for wild-type andmutant TbpBs

Protein Mutation Loop Kd MethodApH49 TbpB WT 55 nM ITCApH49 TbpB F171A L8 NDB ITCApH49 TbpB WT 44 nM SPRApH49 TbpB F171A L8 TBD SPRApH87 TbpB WT 60 nM SPRApH87 TbpB Y95A L3 585 nM SPRApH87 TbpB Y121A L5 203 nM SPRApH87 TbpB Y174A L8 8.9 uM SPRApH87 TbpB R179E L8 6.1 uM SPRAsH57 TbpB WT 120 nM SPRAsH57 TbpB F63A L1 326 nM SPRAsH57 TbpB F152A L5 495 nM SPRHp5 TbpB WT 21 nM BLIHp5 TbpB Y93 A L3 TBD BLIHp5 TbpB Y117A L5 TBD BLIHp5 TbpB(e4535)

Y167A L8 40uM BLI

Hp5 TbpB W176A L8 TBD BLIHp5 TbpB(e4597)

Y167A,W176A

L8 NDB BLI

TBD – To Be DeterminedNDB – No Detectable BindingITC – isothermal titration calorimetrySPR – surface plasmon resonancesBLI – biolayer interferometry

FIGURE 22

Days of Survival

15

6

12

Control Native TbpB

Mutant TbpB

Porcillis Glasser

9

3

Mild or no symptoms or pathologyModerate to severe symptoms and pathology

Survival: Clinical Symptoms:

Transferrin Receptor-‐based Vaccine

Engineered mutant TbpB is a superior antigen

Mutant TbpBNative TbpBPorcillis GlasserControl

0 24 72 96 120 144 168 312 33648

Hours after challenge

% Survival

20

40

60

80

100

CEAC

AM1

infecteduninfected

wildtype

A B

huCEACAM1 N. meningitidis

Colonization of Humanized Transgenic Mice

DAPI

Scott Gray-‐Owen

Men C Conjugate Vaccine

Prevents ColonizationTbpB and TbpB C-‐lobe

Prevents Colonization

Factor H binding protein protects against sepsis but not colonization

PorA protects against sepsis but not colonization.

NHBA partially protects against sepsis but not colonization

NadA protects against sepsis and colonization

Prevention of Colonization is not a Common Feature of Protein Antigens

Invasive disease challenge with H44/76FHbp match

0 12 24 36 480

25

50

75

100

Time (h)

Per

cent

sur

viva

l

AdjuvantBexsero

Invasive disease challenge with SK016PorA match

0 12 24 36 480

25

50

75

100

Time (h)

Per

cent

sur

viva

l

AdjuvantBexsero

Invasive disease challenge with S3446NHBA match

0 12 24 36 480

25

50

75

100

Time (h)

Per

cent

sur

viva

l

AdjuvantBexsero

Invasive disease challege with BuFa 02/03NadA match

Time (h)

Per

cent

sur

viva

l

0 12 24 36 480

25

50

75

100AdjuvantBexsero

Bexse

ro

Adjuva

nt

10

100

1000

detection limit

CFU

per

ani

mal

3 day colonization challenge with H44/76FHbp match

Bexse

ro

Adjuva

nt

10

100

1000

detection limit

CFU

per

ani

mal

3 day colonization challenge with SK016PorA match

Bexse

ro

Adjuva

nt

10

100

1000

detection limit

CFU

per

ani

mal

3 day colonization challenge withS3446NHBA match

Bexse

ro

Adjuva

nt

10

100

1000

detection limit

CFU

per

ani

mal

3 day colonization challenge with BuFa 02/03 APgroup W, NadA match

Engineered TbpB Vaccines

Advantages: • Tf receptors are essential for survival on the mucosal surface (N.

gonorrhoeae, A. pleuropneumoniae) • Engineered TbpB antigens are superior in inducing a protective

immune response in the native host (H. parasuis challenge experiments)

• TbpB (and TbpB C-‐lobe) are capable of preventing colonization in a humanized mouse model (unlike antigens from commercial vaccines)

• Surrogate host-‐pathogen systems can provide proof of concept Question:

• Could a TbpB-‐based vaccine be used eradicate receptor-‐containing bacteria?

TbpB diversity transcends species barriers – single vaccine for the three porcine pathogens.

Phylogenetic groups defined by structural features and interaction with Tf

Cross-‐reactivity analysis predicts small number of engineered antigens required for broad cross-‐protection.

Porcine Pathogen TbpBs Sequence Diversity of TbpBs from Porcine Pathogens

Immunization Experiments in Pigs

From Strain H49

N-‐lobe induces more ‘Group/strain specific’ antibody response.

C-‐lobe induces broadly cross-‐reactive immune response.

Predicts that three representative mutant TbpBs would be capable of inducing a broadly cross-‐reactive and cross-‐protective response.

-‐ Group 3-‐ Group 3-‐ Group 2-‐ Group 1

Meningococcal Vaccine -‐ Sequence/Structural Diversity

Comprehensive evaluation of sequence diversity – intact TbpB & C-lobe.

Two TbpB isotypes (Group 1 vs Group 2-4).

Two antigens selected for immunization experiments (circled in red)

Representative variants selected for analysis of cross-reactivity (arrows).

Paul Adamiak

C-‐lobe Diversity

Intact TbpB Diversity

Sequence Diversity – Evaluation of Cross-‐reactivity

Immunization with TbpBs or C-lobes provides reactivity against diverse representative TbpBs.

Antigen:

Tbp Strain

Jamie Fegan

TbpB-based Meningococcal Vaccine

Can we eliminate important pathogens?

• TbpB present in some commensal Neisseria sp. and in N. gonorrhoeae -‐ potential reservoir for vaccine escape

• overall diversity of Neisseria TbpBs modestly greater than meningococcal TbpBs (i.e. limited number of engineered TbpBs/C-‐lobes required)

• commensal Neisseria with and without TbpB are similar, unlikely to be dependent upon TbpB for iron (eliminating TbpB won’t eliminate commensal Neisseria)

• require systematic approach for evaluating ability to induce complete cross-‐protection -‐ integrated vaccine evaluation pipeline

Global collection of disease isolates Bioinformatics

analysis of antigen diversity

Library of antigenic variants

Structural studies

Bacterial killing (SBA)

+ -‐

URT microbiome Cloning & Expression (SG)

ELISA Assay

immunize

serum

generate strain library

strain library

Engineered antigen

Challenge humanized mice

Integrated Vaccine Evaluation Pipeline

Commercialization

US Provisional Patent Applications

• Dec, 2013 • June, 2014 • October, 2014

PCT Patent Application

• Dec 2014

Exploring Partnerships/Licensing Agreements

Meningococcal Transferrin and Lactoferrin Receptors in

Colonization, Infection and Disease Prevention

ALMA Alberta Livestock

and Meat Agency Ltd.

Development of a Broad-‐Spectrum Porcine Vaccine for Bacterial Respiratory

Infections

University of Passo Fundo (Brazil) Rafael Frandoloso

University of Leon (Spain) Cesar Gutierrez-‐Martin Elias Ferri-‐Rodriguez

Sonia Martinez-‐Martinez

Genome Alberta ALMA

Disease Reduction in Cattle by Elimination of

Colonization by Pathogens

Can$we$eliminate$importantbacterial$pathogens$with ......Human Neisseria meningitidis meningitis,...

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